One of the most important goals in oncology is the identification and elimination of treatment resistant cells; hypoxic cells are the most familiar examples of this type of cell. See, Kennedy, et al., Biochem. Pharm. 1980, 29, 1; Moulder, et al., Int. J. Radioat. Oncol. Biol. Phys. 1984, 10, 695; Adams, Cancer, 1981, 48, 696, all of which are incorporated herein by reference in their entirety. Hypoxic cells are seldom found in normal tissues, and are generally found only in conjunction with certain tumors, vascular diseases, wounded tissue, or after a stroke.
As certain tumors enlarge, the tissue often outgrows its oxygen and nutrient supply because of an inadequate network of functioning blood vessels and capillaries. Although the cells deprived of oxygen and nutrients may ultimately die, at any given time a tumor may produce viable hypoxic cells. These hypoxic cells, although alive, have very low oxygen concentrations because of their remoteness from the blood vessels.
The level of molecular oxygen has important implications in disease diagnosis and prognosis. In medical oncology, for example, hypoxic cells in solid tumors may be highly resistant to killing by some forms of chemotherapy. When chemotherapeutic agents are administered to patients, the agents are carried through the functioning blood vessels and capillaries to the target tissue. Because hypoxic tissue lacks a fully functioning blood supply network, the chemotherapeutic drugs may never reach the hypoxic cells; instead, intervening cells scavenge the drug. The result is that the hypoxic cells survive and recurrence of the tumor is possible. Kennedy, et al., supra.
Tissue hypoxia also hinders the effectiveness of radiation therapy against tumors. Radiation treatment is most effective in destroying oxygen containing cells because oxygen is an excellent radiation sensitizer. The presence of hypoxic cells impedes this treatment because their low oxygen concentration renders the ionizing radiation relatively ineffective in killing the cancerous cells. Therefore, hypoxic cells are more likely to survive radiation therapy and eventually lead to the reappearance of the tumor. The importance of hypoxic cells in limiting radiation responsiveness in animal tumors is well known, Adams, supra; Moulder, et al., supra; Chapman, et al., “The Fraction of Hypoxic Clonogenic Cells in Tumor Populations,” in Biological Bases and Clinical Implications of Tumor Radioresistance 61, G. H. Fletcher, C. Nevil, & H. R. Withers, eds., 1983. Studies have revealed that such resistant cells greatly affect the ability of radiation and chemotherapy to successfully sterilize tumors in animals. Substantial work since that time has shown similar problems in human tumors. Despite the progress in animal studies regarding the identification of hypoxic cells, limited success has been achieved in humans. One reason for this disparity may relate to differences in tumor growth and other host related factors, but in addition, there has been no suitably accurate method to assess tissue oxygen at a sufficiently fine resolution.
Venous oxygen pressure is generally ˜35 Torr, an oxygen level providing nearly full radiation sensitivity. As the oxygen level decreases below 35 Torr, radiation resistance gradually increases, with half-maximal resistance at about 3.5 Torr, and full resistance at about 0.35 Torr. Therefore, it is necessary to measure much lower oxygen levels than are usually encountered in normal tissue. Current technology does not meet this need.
Nitroheterocyclic drugs have been under extensive investigation as hypoxia markers. It is known that this class of compounds can provide sufficient sensitivity to monitor the low oxygen partial pressures described above. This technique involves the administration of nitroaromatic drugs to the tissue of interest. The drugs undergo bioreductive metabolism at a rate which increases substantially as the tissue's oxygen partial pressure decreases. The result of this bioreductive metabolism is that reactive drug products are formed which combine chemically to form adducts with predominantly cellular proteins. Because the metabolic binding of these compounds to cellular macromolecules is inhibited by oxygen, these compounds bind to hypoxic cells in preference to normal, healthy, oxygen-rich tissue. This preferential metabolic binding, or adduct formation, provides a measure of the degree of hypoxia. Koch, et al., Int. J. Radiation Oncology Biol. Phys., 1984,10, 1327.
Misonidazole (MISO) 3-methoxy-1-(2-nitroimidazol-1-yl)-2-propanol and certain of its derivatives have been under extensive investigation as indicators of hypoxia in mammalian tissue. Chapman, et al., Int. J. Radiat. Oncol. Biol. Phys., 1989,16, 911; Taylor, et al., Cancer Res., 1978, 38, 2745; Varghese, et al., Cancer Res., 1980, 40, 2165. The ability of certain misonidazole derivatives to form adducts with cellular macromolecules, referred to as binding throughout this application, has formed the basis of various detection methods.
For example, 3H or 14C labeled misonidazole has been used in vitro and in vivo, with binding analyzed by liquid scintillation counting or autoradiography. Chapman, 1984 supra; Urtasun, 1986, supra; Franko, et al., Cancer Res., 1987, 47, 5367. A monofluorinated derivative of misonidazole has utilized the positron emitting isotope 18F for imaging bound drug in vivo, Rasey, et al., Radiat. Res., 1987, 111,292. The method of the preparation of the PET derivative of ethanidazole was described in Tewson T. J., Nuclear Medicine & Biology, 1997 24(8):755–60. An iodine isotope has been incorporated into another azomycin derivative, azomycin arabinoside, allowing radiology techniques of detection. Parliament, et al., Br. J. Cancer, 1992, 65, 90.
A hexafluorinated derivative of misonidazole 1-(2-hydroxy-3-hexafluoro-isopropoxy-propyl)-2-nitroimidazole has been assayed directly (no radioactive isotopes) via nuclear magnetic resonance spectroscopy (NMR or MRI) techniques. Raleigh, et al., Int. J. Radiat. Oncol. Biol. Phys., 1984, 10, 1337. Polyclonal antibodies to this same derivative have allowed immunohistochemical identification of drug adducts. Raleigh, et al., Br. J. Cancer, 1987, 56, 395.
The bioreductive drug assays described above do not directly measure oxygen partial pressure, even though this is the required value, using the example of radiation therapy to predict radiation response. Rather, the assays measure adduct formation, a biochemical process which is inhibited by oxygen. The data generated using these methods has shown that the degree of inhibition by oxygen varies substantially from tissue to tissue. Franko, et al., 1987, supra. Furthermore, the maximum rate of adduct formation in the complete absence of oxygen is also highly variable from tissue to tissue, as is the maximum percentage of inhibition by oxygen, Koch, in Selective Activation of Drugs by Redox Processes, Plenum Press, pp. 237–247, Adams, et al., eds, New York, 1990. Another way of expressing these limitations is that the bioreductive formation of nitroaromatics provides only a relative indication of varying oxygen levels, but is inadequate at providing an absolute measurement of oxygen partial pressure because there are several factors which affect adduct formation in addition to changes in oxygen, non-oxygen-dependent factors. Additionally, the choice of nitroaromatic drug affects the variability related to the non-oxygen-dependent factors.
Early research efforts (i.e., before the invention claimed in U.S. Pat. No. 5,540,908 on Nov. 19, 1992) had focused on misonidazole and certain of its derivatives. However, misonidazole is the most susceptible of several drugs tested to non-oxygen-dependent variations in adduct formation. Koch, Selective Activation, supra. Other problems relate to various physicochemical properties of existing drugs, all of which can influence the non-oxygen dependent variations in adduct formation. For example, the hexafluorinated misonidazole derivative described above had a high degree of insolubility.
Thus, we have focused our previous study on a 2-nitroimidazole which has greatly superior properties to misonidazole for the purpose of hypoxia detection This drug is 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide (hereinafter referred to as EF5), and 2-(2-nitro-1H-imidazol-1-yl)-N-(3,3,3-trifluoropropyl) acetamide (hereinafter referred to as EF3), see, U.S. Pat. No. 5,540,908, issued to Koch et al., the disclosure of which is herein incorporated by reference in its entirety, as well as (N-(3-fluoropropyl)-2-(2-nitroimidazol-1[H]-yl)-acetamide (EF1), see U.S. Ser. No. 09/123,300, also incorporated herein by reference and assigned to the same entity. Our previous studies have employed monoclonal antibodies to detect the adducts of EF3 and EF5.
Incorporation of 18F into 2-nitroimidazole compounds provides an opportunity to use these agents for the detection of hypoxia by positron emission tomography (PET). See, Jerabek, et al., Applied Radiation & Isotopes, 1986 37 (7), 599–605; see, Mathias et al., “Radiolabelled hypoxic cell sensitizers: tracers for assessment of ischemia,” Life Sciences, 1987 41 (2), 199–206. Several groups have developed 18F-labeled nitroimidazole-based PET assays, for example, [18F]-fluormisonidazole. See, Rasey et al, Radiation Research, 1987 111, (2),292–304; Rasey et al., Int'l J. of Rad. One., Bio., Phys., 1996 36(2),417–428; Grierson, Journal of Nuclear Medicine, 1989 30 (3), 343–50; Koh, et al., International Journal of Radiation Oncology, Biology, Physics, 1992 22 (1), 199–212; [18F]-fluoroerythronitroimidazole, See, Yang, et al., Radiology, 1995 194 (3), 795–800; and, [18F]-fluoroetanidazole, See, Tewson, Nuclear Medicine & Biology, 1997 24 (8), 755–60.
The first described and most investigated compound of this type is [18F]-fluoromisonidazole. This agent has been studied in several anatomic sites in humans including gliomas, see, Valk, et al. Journal of Nuclear Medicine, 1992 33 (12), 2133–7; lung cancer, see, Koh, et al., Acta Oncologica, 1994 33 (3),323–7; and nasopharyngeal carcinoma, see, Yeh, et al, European Journal of Nuclear Medicine, 1996 23 (10), 1378–83. However, despite the extensive investigations, none of these currently developed compounds is accepted clinically as a PET marker of hypoxia. For example, it has been shown that [18F]-fluoromisonidazole is not stable in vivo, and produces multiple radioactive products distinct from the parent drug following renal clearance. See, Rasey, et al., Journal of Nuclear Medicine, 1999 40 (6), 1072–9. Our goal, therefore, has been to employ all the other beneficial aspects of hypoxia detection by EF5, including high drug stability in vivo, ability to cross blood-brain barrier, etc., with non-invasive detection of 18F incorporated into its molecular structure.
Recently, [18F]-EF1 compounds have been developed as PET hypoxia markers. This compound was synthesized using nucleophilic substitution of the bromine atom of a precursor 2(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)-acetamide by [18F]-F-. See, Kachur et al., Journal of Applied Radiation and Isotopes, 1999, 51 (6), 643–650. [18F]-EF1 has shown good potential for labeling of hypoxic tumors and a relatively uniform biodistribution limited by slow equilibration with brain tissue Evans, et al., Journal of Nuclear Medicine, 2000 Vol. 41, 327–336. As EF5 has been shown to predict radiotherapy resistance in individual rodent tumors with well documented pharmacological properties, attempts were made to label this compound with 18F for use in non-invasive imaging techniques. Until now, attempts to incorporate 18F into a site already containing other fluorine atoms have been unsuccessful. Thus, a need exists for new methods of incorporating 18F labels into compounds that are useful in non-invasive imaging techniques, such as PET.